Yuet Wai Kan

An important function of Nrf2 in combating oxidative stress:Detoxification of acetaminophen

Nrf2, a member of the “cap ‘n collar” group of transcription factors, is important for protecting cells against oxidative damage. We investigated its role in the detoxification of acetaminophen [N-acetyl-p-aminophenol (APAP)]-induced hepatotoxicity. When Nrf2 knockout (Nrf2−/−) and wild-type mice were given APAP by i.p. injection, the Nrf2−/− mice were highly susceptible to APAP treatment. With doses of APAP that were tolerated by wild-type mice, the Nrf2−/− mice died of liver failure. When hepatic glutathione was depleted after a dose of 400 mg/kg of APAP, the wild-type mice were able to compensate and regain the normal glutathione level. In contrast, the glutathione level in the Nrf2−/− mice was not compensated and remained low. This was because of the decrease in the gene expression of gcsH and gcsL as well as gss in the livers of the Nrf2−/− mice. In addition, the expression of ugt1a6 and gstpi that detoxify APAP by conjugation was also decreased. This increased susceptibility of the Nrf2−/− mice to APAP, because of an impaired capacity to replenish their glutathione stores, compounded with a decreased detoxification capability, highlights the importance of Nrf2 in the regulation of glutathione synthesis and cellular detoxification processes.

Identification of the NF-E2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis

The antioxidant responsive element (ARE) mediates transcriptional regulation of phase II detoxification enzymes and antioxidant proteins such as NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferases, and glutamate-cysteine ligase. In this study, we demonstrate that NF-E2-related factor-2 (Nrf2) plays a major role in transcriptional activation of ARE-driven genes and identify Nrf2-dependent genes by oligonucleotide microarray analysis using primary cortical astrocytes from Nrf2+/+ and Nrf2−/− mice. Nrf2−/−astrocytes had decreased basal NQO1 activity and no induction bytert-butylhydroquinone compared with Nrf2+/+ astrocytes. Similarly, both basal and induced levels of human NQO1-ARE-luciferase expression in Nrf2−/− astrocytes were significantly lower than in Nrf2+/+ astrocytes. Furthermore, human NQO1-ARE-luciferase expression in Nrf2−/−astrocytes was restored by overexpression of Nrf2, whereas ARE activation in Nrf2+/+ astrocytes was completely blocked by dominant-negative Nrf2. In addition, we observed that Nrf2-dependent genes protected primary astrocytes from H2O2- or platelet-activating factor-induced apoptosis. In support of these observations, we identified Nrf2-dependent genes encoding detoxification enzymes, glutathione-related proteins, antioxidant proteins, NADPH-producing enzymes, and anti-inflammatory genes using oligonucleotide microarrays. Proteins within these functional categories are vital to the maintenance and responsiveness of a cell defense system, suggesting that an orchestrated change in gene expression via Nrf2 and the ARE gives a synergistic protective effect against oxidative stress.

Nrf2-mediated neuroprotection in the MPTP mouse model of Parkinson's disease: Critical role for the astrocyte

Oxidative stress has been implicated in the etiology of Parkinsons disease (PD) and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of PD. It is known that under conditions of oxidative stress, the transcription factor NF-E2-related factor (Nrf2) binds to antioxidant response element (ARE) to induce antioxidant and phase II detoxification enzymes. To investigate the role of Nrf2 in the process of MPTP-induced toxicity, mice expressing the human placental alkaline phosphatase (hPAP) gene driven by a promoter containing a core ARE sequence (ARE-hPAP) were used. ARE-hPAP mice were injected (30 mg/kg) once per day for 5 days and killed 7 days after the last MPTP injection. In response to this design, ARE-dependent gene expression was decreased in striatum whereas it was increased in substantia nigra. The same MPTP protocol was applied in Nrf2+/+ and Nrf2−/− mice; Nrf2 deficiency increases MPTP sensitivity. Furthermore, we evaluated the potential for astrocytic Nrf2 overexpression to protect from MPTP toxicity. Transgenic mice with Nrf2 under control of the astrocyte-specific promoter for the glial fribillary acidic protein (GFAP-Nrf2) on both a Nrf2+/+ and Nrf2−/− background were administered MPTP. In the latter case, only the astrocytes expressed Nrf2. Independent of background, MPTP-mediated toxicity was abolished in GFAP-Nrf2 mice. These striking results indicate that Nrf2 expression restricted to astrocytes is sufficient to protect against MPTP and astrocytic modulation of the Nrf2-ARE pathway is a promising target for therapeutics aimed at reducing or preventing neuronal death in PD.

Nrf2 transcription factor, a novel target of keratinocyte growth factor action which regulates gene expression and inflammation in the healing skin wound

ABSTRACT Keratinocyte growth factor (KGF) is a potent mitogen for epithelial cells, and it promotes survival of these cells under stress conditions. In a search for KGF-regulated genes in keratinocytes, we identified the gene encoding the transcription factor NF-E2-related factor 2 (Nrf2). Nrf2 is a key player in the cellular stress response. This might be of particular importance during wound healing, where large amounts of reactive oxygen species are produced as a defense against invading bacteria. Therefore, we studied the wound repair process in Nrf2 knockout mice. Interestingly, the expression of various key players involved in wound healing was significantly reduced in early wounds of the Nrf2 knockout animals, and the late phase of repair was characterized by prolonged inflammation. However, these differences in gene expression were not reflected by obvious histological abnormalities. The normal healing rate appears to be at least partially due to an up-regulation of the related transcription factor Nrf3, which was also identified as a target of KGF and which was coexpressed with Nrf2 in the healing skin wound. Taken together, our results reveal novel roles of the KGF-regulated transcription factors Nrf2 and possibly Nrf3 in the control of gene expression and inflammation during cutaneous wound repair.

ANTENATAL DIAGNOSIS OF SICKLE-CELL ANÆMIA BY D.N.A. ANALYSIS OF AMNIOTIC-FLUID CELLS

The polymorphism of a restriction endonuclease site has been used for antenatal diagnosis of sickle-cell disease. In a normal person, the beta-globin gene was contained in a Hpa I-digested D.N.A. fragment 7.6 kilobases (kb) in length. In a family where the sickle gene was contained in a variant 13.0 kb fragment, restriction endonuclease mapping was used for antenatal diagnosis. The D.N.A. from amniotic-fluid cells produced both the 7.6 and the 13.0 bk beta-globin gene fragments, indicating the diagnosis of sickle-cell trait. This confirmed the diagnosis reached after investigation of a 100% sample of fetal blood. The method is sensitive and can be performed with cells obtained from 15 ml of uncultured amniotic fluid. This approach may prove useful in antenatal diagnosis of other genetic disorders.

Concurrent sickle-cell anemia and α-thalassemia: effect on severity of anemia.

Abstract We studied 47 patients with sickle-cell anemia to determine the effect of α-thalassemia on the severity of their hemolytic anemia. We diagnosed α-thalassemia objectively by using α-globin-gene mapping to detect α-globin-gene deletions, studying 25 subjects with the normal four α-globin genes, 18 with three, and four with two. The mean hemoglobin, hematocrit, and absolute reticulocyte levels (±S.D.) were 7.9±0.9 g per deciliter (4.9±0.6 mmol per liter), 22.9±2.9 per cent, and 501,000±126,000 per cubic millimeter, respectively, in the non-thalassemic group; 9.8±1.6 g per deciliter (6.1±1.0 mmol per liter), 29.0±5.0 per cent, and 361,000±51,000 per cubic millimeter in the group with three α-globin genes; and 9.2±1.0 g per deciliter (5.7±0.6 mmol per liter), 27.5±3.0 per cent, and 100,000±15,000 per cubic millimeter in the group with two α-globin genes. Deletion of α-globin genes was also accompanied by a decreased mean corpuscular hemoglobin concentration (MCHC) in post-reticulocyte erythrocytes and...

Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac

β-thalassemia, one of the most common genetic diseases worldwide, is caused by mutations in the human hemoglobin beta (HBB) gene. Creation of human induced pluripotent stem cells (iPSCs) from β-thalassemia patients could offer an approach to cure this disease. Correction of the disease-causing mutations in iPSCs could restore normal function and provide a rich source of cells for transplantation. In this study, we used the latest gene-editing tool, CRISPR/Cas9 technology, combined with the piggyBac transposon to efficiently correct the HBB mutations in patient-derived iPSCs without leaving any residual footprint. No off-target effects were detected in the corrected iPSCs, and the cells retain full pluripotency and exhibit normal karyotypes. When differentiated into erythroblasts using a monolayer culture, gene-corrected iPSCs restored expression of HBB compared to the parental iPSCs line. Our study provides an effective approach to correct HBB mutations without leaving any genetic footprint in patient-derived iPSCs, thereby demonstrating a critical step toward the future application of stem cell-based gene therapy to monogenic diseases.

Targeted disruption of the ubiquitous CNC-bZIP transcription factor, Nrf-1, results in anemia and embryonic lethality in mice

The CNC‐basic leucine zipper (CNC‐bZIP) family is a subfamily of bZIP proteins identified from independent searches for factors that bind the AP‐1‐like cis‐elements in the β‐globin locus control region. Three members, p45‐Nf‐e2, Nrf‐1 and Nrf‐2 have been identified in mammals. Expression of p45‐Nf‐e2 is largely restricted to hematopoietic cells while Nrf‐1 and Nrf‐2 are expressed in a wide range of tissues. To determine the function of Nrf‐1, targeted disruption of the Nrf‐1 gene was carried out. Homozygous Nrf‐1 mutant mice are anemic due to a non‐cell autonomous defect in definitive erythropoiesis and die in utero.

Impaired liver regeneration in Nrf2 knockout mice: role of ROS‐mediated insulin/IGF‐1 resistance

The liver is frequently challenged by surgery‐induced metabolic overload, viruses or toxins, which induce the formation of reactive oxygen species. To determine the effect of oxidative stress on liver regeneration and to identify the underlying signaling pathways, we studied liver repair in mice lacking the Nrf2 transcription factor. In these animals, expression of several cytoprotective enzymes was reduced in hepatocytes, resulting in oxidative stress. After partial hepatectomy, liver regeneration was significantly delayed. Using in vitro and in vivo studies, we identified oxidative stress‐mediated insulin/insulin‐like growth factor resistance as an underlying mechanism. This deficiency impaired the activation of p38 mitogen‐activated kinase, Akt kinase and downstream targets after hepatectomy, resulting in enhanced death and delayed proliferation of hepatocytes. Our results reveal novel roles of Nrf2 in the regulation of growth factor signaling and in tissue repair. In addition, they provide new insight into the mechanisms underlying oxidative stress‐induced defects in liver regeneration. These findings may provide the basis for the development of new strategies to improve regeneration in patients with acute or chronic liver damage.

Prenatal Diagnosis of β-Thalassemia

Abstract We investigated a method employing synthetic oligonucleotides for the prenatal diagnosis of β-thalassemia due to a single nucleotide mutation. The β0 thalassemia we tested is produced by a...